hsc70 forms a complex with hsp90 and PP1. hsc70 was immunoprecipitated from lysates of 5 × 10⁸ resting platelets with MoAb 1B5 and probed with the indicated antibodies. ³²P-labeling, immunoprecipitations (Ips), gels, and immunoblotting were as described in the Materials and Methods. (A) hsc70 in resting platelets. Preparations were separated by 8% SDS-PAGE minigels and immunoblotted with anti-hsc70 antibody 1B5. Pure hsc70 refers to 5 μg of purified hsc70; lysate refers to a whole-platelet lysate from 1.1 × 10⁷ platelets; IP refers to an anti-hsc70 Ip; and rat IgG refers to a control Ip with nonimmune rat IgG. (B) hsc70 phosphoprotein complex. Both lanes represent anti-hsc70 Ips of ³²P-labeled resting platelets. Western is an anti-hsc70 immunoblot; ³²P is an autoradiogram. (C) Autoradiogram of immunoprecipitates from control (resting) platelets with nonimmune rat and mouse IgGs, and with specific MoAbs against hsc70 (N27, 1B5, and 13D3). Immunoprecipitates were separated by 10% SDS-PAGE minigels. (D) hsc70 complex with hsp90. Both lanes are Ips of resting platelets blotted with anti-hsp90. Anti-hsc70 is an anti-hsc70 Ip; rat IgG is a nonimmune rat IgG Ip. (E) hsc70 complex with PP1. Resting platelet Ips were blotted with antibodies raised against different subunits of PP1. Anti-hsc70 is an anti-hsc70 Ip; rat IgG is a nonimmune rat IgG Ip. The lower labels indicate the antibodies used for blotting. The arrow labels designate specific proteins; PP1C identifies PP1Cα and PP1Cδ, which migrated similarly.